JPH01222768A - Bioreactor - Google Patents

Abstract

PURPOSE:To meet the cell culture of a large scale by providing the bioreactor with the membrane for feeding the culture medium and the other membrane for exchanging the gases using porous hollow fiber membranes which are made hydrophilic by treatment as the membrane for feeding culture medium to make it possible to feed a sufficient amount of oxygen gas to the culture medium and the cells. CONSTITUTION:The culture medium is fed through the membrane 8 and the cells are proliferated on the membrane 8 and the outer space of the membrane 8. In addition, the bioreactor 14 is provided with the other membrane 9 for exchanging gases inside, while the membrane 8 for supplying the medium is constituted with porous hollow fibers which is made hydrophilic by treatment. As a result, sufficient amount of oxygen-containing gas and pH-controlling gas are supplied sufficiently to the culture medium and the cells to grow and proliferate the cells without any trouble whereby the process according the present invention can meet satisfactorily a large scale of cell culture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a bioreactor for growing cells, and more specifically to a bioreactor for culturing cells on a large scale.

[Prior Art] In a culture device using a culture tank for culturing microorganisms or cells, it is known that one way to increase the volumetric efficiency of culture is to increase the supply of aeration gas to the culture medium. In this case, vigorous agitation of the medium is generally performed.

Meanwhile, cells are cultured using a hollow fiber membrane. A culture method called the so-called hollow fiber method is known.

[Problems to be Solved by the Invention] However, in the method of increasing the supply of aeration gas, bubbles come into contact with the cells due to aeration bubbling into the medium, and mechanical stress is applied to the cells due to stirring. Another problem is that cells, especially fragile cells such as animal cells, may be killed or damaged.

On the other hand, in the culture method called the hollow fiber method, the hollow fiber bioreactor and gas exchanger are installed separately, as shown in Figure 3.
Also, there is a problem that the supply of pH adjustment gas is insufficient, making it unsuitable for large-scale cell culture. To explain this point, in a hollow fiber bioreactor,
Although it is necessary to deliver a strictly controlled medium to the cells outside the hollow fiber membrane, the bioreactor 14 and gas exchanger 1
7 is provided separately, as shown in FIG. 3, the pH and Do of the culture medium are measured before (or after) the bioreactor 14 using the PH sensor 13 and the DOO ring 19, and the However, if cells rapidly proliferate, the pH value and Do value of the medium entering the bioreactor 14 will differ. This is because glucose and the like in the medium are consumed by the cells. Therefore, even if the PH value and Do value are measured in front of the bioreactor 14, they will only serve as a guideline, and there is little point in performing gas exchange based on the signals of these values.

Furthermore, in the case of the conventional hollow fiber method, since 02 is supplied only to the medium, there is also the problem that cells become deficient in oxygen and die.

[Means for Solving the Problems] Therefore, the present invention has been made in view of the drawbacks of the prior art described above, and is suitable for large-scale cell culture where 02 gas is supplied not only to the culture medium but also to the cells. The purpose is to provide a suitable bioreactor. According to the present invention, the purpose is to provide a bioreactor in which a culture solution is supplied through a medium supply membrane and cells are grown on the medium supply membrane and in the external space of the medium supply membrane. This can be achieved by a bioreactor characterized in that a membrane for gas exchange is disposed together with the medium supply membrane, and the medium supply membrane is made of a porous hollow fiber membrane treated with hydrophilic Ibi. .

[Function] In the bioreactor of the present invention, a medium is caused to flow inside a medium supply membrane disposed in the bioreactor, and the medium is oozed out to the outside of the medium supply membrane through the pores of the membrane.
The cells implanted on the outside of the medium supply membrane are grown.

At that time, the gas sent through the inside of the gas exchange membrane disposed together with the medium supply membrane in the bioreactor also comes into contact with the medium seeped out from the medium supply membrane through the gas exchange membrane. In addition to supplying 0□ to the culture medium, 0□ is also supplied to the cells implanted outside the culture medium supply membrane.
Supply 2.

Moreover, since the culture medium supply membrane of the present invention has been subjected to a hydrophilic treatment, the amount of water permeation can be increased.

[Example] Hereinafter, the present invention will be explained in detail based on illustrated embodiments.

FIG. 1 is a longitudinal sectional view showing an example of the structure of the bioreactor of the present invention. A culture medium supply 11 is provided inside the cylindrical container 7.
88 and a large number of hollow fiber membranes constituting the gas exchange membrane 9 are arranged, and at both ends of the cylindrical container 7, the inner surface of the cylindrical container 7 and the medium supply membrane 8 and the gas exchange membrane 9 are connected to each other. The outer surface is airtightly supported by a support member lO made of a botting material such as polyurethane resin, and both ends of the culture medium supply membrane 8 and gas exchange membrane 9 are open to both ends of the cylindrical container 7. .

In the cylindrical container 7, the culture medium supply membrane 8 and the gas exchange membrane 9 are arranged such that the gas exchange membrane 9 is disposed in the center and the culture medium supply membrane 8 is disposed outside of the gas exchange membrane 9, and each has a large number of through holes 12 through which gas and liquid can pass. They are separated by a partition wall 11 having a partition wall 11.

In addition, boat 5 is a boat for removing used nutrient medium and cell products from the bioreactor, and boat 6
is an inlet for a sensor that controls the gas supply amount and concentration. Note that the O-ring 19 is provided to maintain airtightness with the outside and to prevent gas and culture medium from mixing.

In the above configuration, the medium A enters the hollow fiber membrane of the medium supply membrane 8 through the medium population 2, and a small amount of medium oozes out to the outside of the medium supply membrane 8 due to the pressure applied to the membrane, and the remaining medium is released from the medium outlet 3 through the tube. exit the shaped container 7. On the other hand, gas B such as air enters the hollow fiber membrane of the gas exchange membrane 9 through the gas population 1, and is supplied to the culture medium through the gas exchange membrane 9 through the through holes 12 of the partition wall 11, and the remaining gas and exchange The gas that is
The liquid then exits the cylindrical container 7.

Culture medium supply membrane 8 constituting the bioreactor of the present invention
The substrate is a porous hollow fiber made of various polymers, such as cellulose acetate,
polysulfone, polyacrylonitrile, fluoropolymer,
Examples include polyolefins (polypropylene, polyethylene, etc.).

The wall of the porous hollow fiber membrane serving as the culture medium supply membrane 8 is provided with a large number of pores that are large enough to allow nutrients, cell wastes, metabolic products, etc. to pass therethrough, but not cells. When the cells themselves are cultured in suspension, the size of the pores will be determined by the size of the cells, but in general, an appropriate average pore size is 10 JLm or less, preferably 8 gm or less.

In addition, since the culture medium supply membrane 8 has been subjected to hydrophilic treatment,
Water permeability can be increased.

Examples of methods for parent tree processing include the following.

On the surface of the hydrophobic porous hollow fiber membrane that is the culture medium supply membrane, carboxymethylethylcellulose, glycerin fatty acid ester, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, poly(2-hydroxyethyl methacrylate), ethylene oxide grafted nylon, 6/66/
Examples include a method of coating with a hydrophilic agent such as 610 nylon terpolymer or 8-type soluble nylon (methoxy nylon).

That is, the culture medium supply membrane 8 has a water permeability coefficient (tJL/m2·
hr·mmHg) is preferably io or more, preferably 100 or more. On the other hand, there is no particular upper limit, but 20.
000 or less, preferably 10,000 or less.

Further, as the culture medium supply membrane 8, it is also possible to use a membrane, such as an ultrafiltration membrane, which allows small molecular weight compounds such as nutrients and cellular wastes to pass through, but does not allow large molecular weight compounds to pass through.

Gas exchange 11 constituting the bioreactor of the present invention
99 may be made of various polymers, such as cellulose, polyacrylonitrile, polycarbonate, polyvinylidene fluoride, polymethyl methacrylate, and polyethylene.

Examples include polypropylene, silicone rubber, and modified materials thereof.

Further, the gas exchange membrane 9 needs to have gas permeability. That is, the gas exchange membrane 9 has a gas permeability coefficient (m /
■2・hr・mmHg) is 10 or more, preferably 100
It is advantageous that the above is the case.

Further, as the gas exchange membrane 9, a hollow fiber membrane is preferable because it can have a small membrane area per unit volume. In particular, porous membranes with high permeability are preferred, and polyolefin porous membranes are particularly preferred.

In addition, in FIG. 1, for convenience of explanation, the shapes are concentric,
A gas exchange membrane 9 is provided in the center, and a culture medium supply membrane 8 is provided on the outside thereof, separated by a partition wall 11. However, in order to sufficiently supply gas to the culture medium, it is necessary to A configuration in which the exchange membrane 9 and the culture medium supply membrane 8 are mixed is more preferable.

In addition, in order to proliferate cells, it is absolutely necessary to eliminate bacterial contamination, so a polypropylene porous membrane is used as the gas exchange membrane 9, a polyethersulfone ultrafiltration membrane is used as the medium supply membrane 8, and a cylindrical It is more preferable to use a polycarbonate container as the container 7 and a polyurethane resin as the support member 10 so that the entire bioreactor can be sterilized with high temperature and high pressure steam.

FIG. 2 shows a cell culture system using the bioreactor of the present invention, which includes a bioreactor 14 equipped with a pH sensor 13 and a Do (dissolved oxygen) sensor 18, a medium tank 15, a pH sensor, and a flow rate of perfusate. This figure shows a system consisting of a pump ld that controls the pump ld. On the other hand, FIG. 3 shows a conventional cell culture method using a bioreactor and a gas exchanger. The difference from FIG. 2 is that the gas exchanger 17 is not included in the bioreactor 14, and The point is that it is installed outside.

Therefore, the results of implementing the present invention based on FIGS. 2 and 3 will be described in more detail below.

(Example) As a culture medium supply membrane, the inner diameter is 300 pm and the outer diameter is 4001 Lm.
, 1142.500 polypropylene porous hollow fibers coated with glycerin fatty acid ester, with an average pore diameter of 0.3 gm and a porosity of 68%, and an inner diameter of 30 as a gas exchange membrane.
Polypropylene porous hollow fiber JIQ2,5 with 0JLm, outer diameter 400pm, average pore diameter 0.21Lm, porosity 65%
A bioreactor consisting of 0.00 was used. (Effective membrane area is 0.5 m'' for both.) Bioreactor 14 and PH sensor 13, DO (
dissolved oxygen) sensor 18, CO□ sensor (not shown)
The culture medium tank 15 and pump 16 were connected with silicone tubes to form a closed circuit. The inside of the circuit was brimmed, and the entire circuit was sterilized using high-pressure steam for 25 minutes.

After sterilization, the circuit was placed in a thermostat at 37°C, and serum-free Eagle's ME was used as the basal medium.
M-E (Minimum Es5nential Med
ium-Eagle) medium (containing penicillin potassium 10,000 units/L and kanamycin sulfate 100 mg/L) at 80+sJL/sin for 48 hours, 10%
FBS (Fetal Bovine Serum) (
The medium was replaced with MEM-E medium containing (calf serum).

HeLa ()IeLa) cells were placed 5x in the space outside the hollow fiber membrane.
106 cells/mJ1 were inoculated. After inoculation, the bioreactor was rotated 90 degrees every hour without circulating the medium for 4 hours to uniformly disperse HeLa cells in the space outside the hollow fiber membrane. After that, the culture medium was incubated at 40 vs/■in, 4
After circulating for an hour, the flow rate was increased to 80 sl/in. Similarly, to maintain the pH of the cell growth medium at 7.4, air (1000cc/i+in) and carbon dioxide gas (50cc
c/win) flowed into the inside of the gas exchange membrane.

The medium of No. 29 was exchanged every 3 days for 15 days, and the glucose concentration, oxygen partial pressure, and carbon dioxide gas partial pressure in the medium tank were measured.

On the 166th day, the medium was replaced every two days, and the apparatus was stopped after 31 days.

After stopping the device, remove PBS (phosphate buffer) in the medium supply membrane.
(-), 0.25% trypsin, each 1
After culturing at 37°C for 5 minutes, the cells were collected by suspension and the number of cells was calculated. Similarly, the inside of the medium supply membrane was filled with PBS (-
), sequentially replaced with 2% geltaraldehyde, 2.5
After fixing with % glutaralhyde overnight, PBS
After washing with water (-), staining with 1% osnic acid, freeze-drying was performed, and after gold deposition, SEM (scanning electron microscope) observation was performed.

The glucose concentration of the medium was adjusted to an initial value of 100 g/dJ1. No significant decrease in glucose concentration was observed for 6 days after the start of culture (medium exchanged twice).

From day 9, a gradual decrease in glucose concentration was observed, and 155
Day 1 decreased from 100-g/di to 20-g/d over 3 days. From then on, measurements taken every two days from the 166th day showed that 1
There was a nearly constant decrease from 00 g/d to 30 yag/di.

Oxygen partial pressure and carbon dioxide partial pressure were 150.20m from beginning to end.
Hg is almost constant, and pH fluctuates from 7.36 to 7.
．． It remained stable between 40 and 40.

The number of cells cultured for 31 days is 6X10' cells.
/tafL.

Observation by SEM shows that the cells attached to the hollow fiber membrane are
It was growing inside the membrane, and also growing outward. Cells that had entered the inside of the membrane did not show much three-dimensional growth, but in cells that grew toward the outside of the membrane, the cells were adjacent to each other, forming a three-dimensional structure formed in vivo. showed similar growth.

(Comparative example) Using the same culture medium supply membrane and gas exchange membrane as used in the example, a bioreactor and a gas exchanger each having an effective membrane area of 0.5 m2 were used separately as shown in Figure 3. did.

Sterilization, briming, and cell culture were performed in the same manner as in Examples.

As a result, the glucose concentration gradually decreased from the 9th day as in the example, but the glucose concentration did not change from the initial value of 100 vsg/dfL from the 155th day.

Furthermore, the pH fluctuated wildly between 5.83 and 8.14, and the oxygen partial pressure also fluctuated wildly.

The glucose concentration did not change from day 15 onwards because the cells had died, and when the oxygen partial pressure inside the bioreactor was measured, it was mostly 0.

This is presumed to be because the dissolved oxygen in the medium in the bioreactor was consumed as the cells proliferated, and as a result, the cells died due to almost no oxygen remaining.

[Effects of the Invention] As explained in detail above, according to the bioreactor of the present invention, a gas exchange membrane is disposed in the bioreactor together with a culture medium supply membrane, and a porous membrane treated to make it hydrophilic is used as the culture medium supply membrane. Since a hollow fiber membrane is used, 02 gas and pH adjustment gas are sufficiently supplied to the culture medium and cells, allowing cell growth and proliferation without any hindrance, and is fully compatible with large-scale cell culture. It has the advantage of

[Brief explanation of the drawing]

Fig. 1 is a vertical cross-sectional view showing an example of the structure of the bioreactor of the present invention, Fig. 2 is a schematic diagram showing a cell culture method using the bioreactor of the present invention, and Fig. 3 is a conventional bioreactor and a gas FIG. 1 is a schematic diagram showing a cell culture method using an exchanger. 7... Cylindrical container, 8... Culture medium supply membrane, 9... Gas exchange □ membrane, 10... Support member, 11-... Partition wall, 12
-...Through hole, 13--pH sensor, 14--Bioreactor, 17--Gas exchanger, 18-DO sensor.

Claims (1)

[Claims] (1) In a bioreactor in which a culture solution is supplied through a medium supply membrane and cells are grown on the medium supply membrane and in a space outside of the medium supply membrane, the medium supply membrane is used for gas exchange together with the medium supply membrane in the bioreactor. 1. A bioreactor, characterized in that the membrane is provided with a membrane, and the culture medium supply membrane is made of a porous hollow fiber membrane that has been subjected to a hydrophilic treatment.